Tumor necrosis factor .alpha. (TNF.alpha.) is a cytokine primarily produced by activated macrophages. TNF.alpha. stimulates T-cell and B-cell proliferation and induces expression of adhesion molecules on endothelial cells. This cytokine also plays an important role in host defense to infection.
TNF.alpha. activities are mediated through two distinct receptors, TNFR-p55 and TNFR-p75. These two receptors also mediate activities triggered by soluble lymphotoxin .alpha. (LT-.alpha.) secreted mainly by activated lymphocytes. Specific stimulation of TNFR-p55 induces TNF activities such as in vitro tumor cell cytotoxicity, expression of adhesion molecules on endothelial cells and keratinocytes, activation of sphingomyelinase with concomitant increases of ceramide, activation of NF-.kappa.B and induction of manganese superoxide dismutase mRNA. Specific stimulation of TNFR-p75 results in proliferative response of mouse and human thymocytes and cytoxic T cells, fibroblasts and natural killer cells and in GM-CSF secretion in PC60 cells.
TNF, especially in combination with .gamma.-interferon (IFN-.gamma.), has the ability to selectively kill or inhibit malignant cell lines that is unmatched by any other combination of cytokines. Clinical trials in cancer patients with TNF-.alpha. antitumor therapy have been disappointing, however, because the toxic side effects of TNF have prevented obtaining effective dose levels in man. These toxic side effects have been attributed to TNF binding to the TNFR-p75 receptor while the cytotoxic activity on malignant cells has been attributed to binding of TNF to the TNFR-p55 receptor.
TNFR-p55 and TNFR-p75 from human (Loetscher et al., "Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor", Cell 61:351-359, 1990; Gray et al., "Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein", Proc. Natl. Acad. Sci. USA 87:7380-7384, 1990; Schall et al., "Molecular cloning and expression of a receptor for tumor necrosis factor", Cell 61:361-370, 1990; Smith et al., "A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins", Science 248:1019-1023, 1990) and mouse (Goodwin et al., "Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor", Mol. Cell Biol. 11:3020-3026, 1991); have been cloned. Random and site-specific mutagenesis have shown that amino acids involved in receptor binding of TNF are located at each interface between two subunits, particularly loops 29-36, 84-91, and 143-148. Loetscher et al., "Human Tumor Necrosis Factor .alpha.(TNF.alpha.) Mutants with Exclusive Specificity for the 55-kDA or 75-kDa TNF Receptors", J. Biol. Chem., 268(35):26350-26358, (1993) reported that generally, mutations in the loop from position 29 to 34 and at positions 86 and 146 preferentially impaired binding to the 75-kDa TNF receptor, while mutations in the region from 143 to 145 mainly affected binding to the 55-kDa TNF receptor.
Two TNF mutants, L29S and R32W, which have been characterized in detail, have maintained almost completely the ability to bind TNFR-p55, while the TNFR-p75 affinity is decreased drastically (Van Ostade et al., "Human TNF mutants with selective activity on the p55 receptor", Nature 361:266-268, 1993). Van Ostade et al. also found that charge reversal at position 146 (E146K) resulted in a marked differential binding and activity. The R32W and E146K mutants were also shown to have decreased activity toward neutrophils and endothelial cells, two cells type believed to play an important rule in TNF-induced systemic toxicity. Thus, it was suggested that the use of TNF-R55-specific mutants, L29S, R32W and E146K in cancer patients may result in reduced general toxicity. A reversed differential binding affinity for TNFR-p75 was less affected as compared to TNFR-p55, and concomitant receptor-mediated activity has also been observed with several substitutions at position 143 in human TNF.
Zhang et al., "Site-directed Mutational Analysis of Human Tumor Necrosis Factor-.alpha. Receptor Binding Site and Structure-Functional Relationship" J. Biol. Chem. 267(33):24069-24075 (1992) examined the structure and functional relationship of site-directed mutation of TNF on receptor binding and found the loop containing Tyr.sup.87 of the TNF molecule to be the most likely to make direct contact with the TNF receptor. It is suggested that since Tyr.sup.87 may form a hydrogen bond with Ala.sup.33, proline substitution at nearby residues on the same loop as Ala.sup.33 could impair the normal interaction of Ala.sup.33 with Tyr.sup.87 by altering the backbone conformation, leading to impaired receptor binding. The effect of mutation at the Ala.sup.33 position was not examined in these experiments.
It has now been found that conservative mutations of the alanine at position 33 does not impair receptor binding to TNFR-p75, but rather results in mutant human tumor necrosis factor-.alpha. proteins which retain full or near full capability to bind to the TNFR-p75 receptor while retaining only a limited capability to bind the TNFR-p55 receptor. These mutant tumor necrosis factor proteins, if acting as TNFR-p75 antagonists, are useful in inhibiting systemic toxicity induced by TNF. On the other hand, if these mutant TNF-.alpha. proteins act as TNFR-p75 agonists, they are useful as TNFR-p75 specific desensitizers at lower doses which suppress systemic toxicity. These mutant proteins are useful as immune stimulators due to their ability to promote lymphocyte proliferation.